The long term objective of this proposal is to illustrate the molecular mechanism of aponecrosis. Aponecrosis is defined as caspase-independent cell death induced by the death receptors such as Fas and TNFalpha receptors. Aponecrotic cell death, which occurs in certain cell types when induced with Fas or TNFalpha in the presence of pan caspase inhibitors, lacks the typical apoptotic feature such as cytoplasm and nuclear condensation and DNA cleavage, and is not associated with caspase activation. Instead, aponecrotic cell death exhibit nuclear and cytoplasm swelling which is typically associated with necrosis. To illustrate this receptor-mediated caspase-independent necrosis pathway, we developed a high throughput screen for small molecule inhibitors of caspase-independent cell death. Such screens allowed us to identify three small molecules, MTHtrp, U2 and U3, which effectively block Fas-mediated caspase-independent cell death. Specifically, MTHtrp blocks TNF/zVAD and Fas/zVAD induced cell death in all cell types tested; whereas U2 blocks TNF/zVAD and Fas/zVAD induced cell death only in certain cell types. Thus, MTHtrp may define a common regulator of necrosis whereas U2 defines a cell -type/pathway-specific control point. This proposal is to use cellular, molecular and chemical genetic approaches to characterize the molecular pathway of aponecrosis. This work is a close collaboration between John Porco, an organic chemist at Boston University, and Junying Yuan, a cell biologist at Harvard Medical School. The Specific Aim 1 is to evaluation of the roles of Bcl-xL, caspases and stress kinases in necrotic cell death. Antiapoptotic members of the Bcl-2 family inhibit apoptosis by preventing mitochondrial damage; but their roles in aponecrosis are not well characterized. Our preliminary data indicate that caspases may normally act to inhibit the caspase- independent necrosis pathway. We would determine the identity of the caspases involved by expressing cellular caspase inhibitors, dominant negative mutants and caspase knockout EF cells. We showed in our preliminary results that p38 may be a cell type/signal-specific necrosis mediator. We would like to further characterize the roles of p38 and other stress kinases in regulating aponecrosis. The Specific Aim 2 is to use chemical genetic approach to identify the key molecules involved in regulating aponecrosis. We would synthesize affinity reagent of MTHtrp to identify its cellular target involved in regulating aponecrosis. We would use parallel synthesis to generate derivatives of MTHtrp to identify more effective inhibitors of aponecrosis. As an alternative approach, we would generate derivatives of U2 using combinatorial method to develop better cell type/pathway-specific inhibitors of necrosis and synthesize U3 to analyze its activity. The Specific Aim 3 is to identify critical mediators of caspase-independent cell death. We will characterize the cellular MTHtrp target. We will test the hypothesis that caspases may cleave certain critical regulators to promote apoptosis and inhibit necrosis. We will analyze the difference in gene expression pattern of normal BalbC3T3 cells and the necrosis-resistant/apoptosis-sensitive subline. Our work may lead to the identification of critical regulators of aponecrosis which plays a similar role as that of caspases in apoptosis.